For Research Use Only. Not for Clinical Use.

Overview of Transferrin

Fig.1 Structure of transferrin.

Transferrin is a highly abundant serum glycoprotein, found in serum at 3-4 mg/mL, which binds iron tightly but reversibly and functions to carry iron to tissues. Transferrin has 679 amino acid residues, is about 80 kDa in size, and possesses two high-affinity Fe³⁺ -binding sites, one in the N-terminal domain and the other in the C-terminal domain. Human transferrin has a half-life reported to be 7-10 days or 10-12 days. The aglycosylated form of human transferrin, which makes up about 2-8% of the total transferrin pool, has a slightly longer half-life of 14-17 days. The extended persistence of transferrin in human serum is due to a clathrin-dependent transferrin receptor-mediated mechanism, which recycles transferrin receptor-bound transferrin back into the circulation. Tf has been developed as a carrier for drug delivery, and it has been proven that Tf fusion proteins have improved pharmacodynamic and pharmacokinetic properties in animals when administered either orally or subcutaneously.

Transferrin Fusion in Half-Life Extension

Fusions of peptides and proteins have been made to human transferrin to the N- and C- termini, as well as to the centrally located hinge region that links the two major lobes of transferrin together. The N terminus of transferrin is free and can be fused directly. The C terminus is more buried and is constrained by a nearby disulfide bond, so flexible linkers are typically used when proteins are fused to the C terminus. This capability was extended by making libraries of peptides against specific targets and then fusing binders from those libraries into aglyco-transferrin (N-terminal, C-terminal, loops, or linker region) to be developed into therapeutic fusion proteins with extended half-lives.

Examples of Transferrin Fusion Application in Half-life Extension

• Diabetes
  The insulinotropic peptides GLP-1 and exendin-4 were genetically fused to non-glycosylated Tf, forming GLP-1-Tf and Ex-4-Tf fusion proteins. Both fusion proteins retained the pharmacological effects of native peptides while extending their length of action. GLP-1-Tf had a half-life of approximately 2 days in cynomolgus monkeys. Proinsulin-transferrin (proINS-Tf), designed by fusing proinsulin to Tf, was converted to active INS-Tf through Tf receptor-mediated endocytosis in hepatic cells by specific cleavage of proinsulin. ProINS-Tf had a delayed but sustained in vivo hypoglycemic effect after s.c. injection and had a 15-fold increase of half-life in diabetic mice.
• Oral bioactivity
  The oral bioactivity of Tf-fusion proteins was confirmed, suggesting that they may be promising candidates for orally delivered therapy. The oral bioactivity of proINS-Tf, expressed from the transgenic rice expression system, was determined in diabetic mice. The results showed that the blood glucose curve of orally administered proINS-Tf at a dose of 800 nmol/kg was similar to an s.c. injection at 22.5 nmol/kg. The exendin-4-Tf fusion protein, expressed in a transgenic tobacco plant, was proven to significantly improve glucose tolerance when administered orally in mice. G-CSF-Tf fusion protein significantly increased the absolute neutrophil counts after oral administration to mice, while G-CSF had no effect. Although no Tf fusion proteins are currently in clinical trials, these observations provide an insight into the potential future development of orally administered fusion proteins based on Tf.