DOI: 10.25549/usctheses-c127-637770
title: Linkers in transferrin fusion proteins: effects on pharmacokinetics and pharmacodynamics

description: Recombinant fusion proteins have become an important class of biomolecules since the invention of recombinant DNA technology. As an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. In the development of recombinant transferrin (Tf)-fusion proteins for protein drug oral delivery, various linkers have been designed to improve the biological activity, or to achieve desired pharmacokinetic and pharmacodynamic properties. ❧ The Introduction in this dissertation first reviewed the mechanism for Tf receptor-mediated protein drug oral delivery, as well as the recombinant Tf-fusion proteins that have been constructed previously in our lab. It also covers the current knowledge of fusion protein linkers and summarizes examples for their applications. The basic function of linkers is to covalently join the functional domains in the fusion proteins.  However, linkers can offer many other advantages for fusion proteins, such as controlled distance between domains, correct folding and confirmation, improved biological activity, increased expression yield, and functional domain separation in vivo. ❧ In order to achieve desired pharmacokinetic profiles and improved biological activity, an in vivo cleavable disulfide linker was designed for in vivo release of protein domains from recombinant Tf-fusion proteins. This novel disulfide linker, based on a cyclopeptide containing a thrombin-sensitive sequence and an intra-molecular disulfide bond, was inserted between Tf and granulocyte colony-stimulating factor (G-CSF)/growth hormone (GH). The fusion proteins linked via the reversible disulfide bond was able to quickly separate the protein domains in vivo upon the reduction of the disulfide bond. After released from the fusion protein, free G-CSF exhibited an improved biological activity in a cell proliferation assay. Due to the short plasma half-life of released free G-CSF, G-CSF-Tf fusion protein with the disulfide linker did not exhibit improved in vivo biological activity compared to its counterpart with a stable peptide linker. This linker design can be adapted to diverse recombinant fusion proteins where in vivo separation of protein domains is required to achieve improved therapeutic effect, desirable pharmacokinetic profile and biodistribution of the functional domains. ❧ Next, recombinant fusion proteins consisting of Tf and GH/G-CSF were constructed as a model for studying the pharmacokinetics (PK) of bifunctional fusion proteins. The impact of linkers on the PK of bifunctional fusion proteins was investigated through the insertion of 3 linkers between the functional domains. The results showed that the insertion of different linkers between the two protein domains altered the binding affinities of the fusion proteins to both domain receptors, and that the fusion proteins’ plasma half-lives were greatly affected. A strong correlation between GH receptor binding affinity and plasma half-life of GH-Tf fusion proteins was observed. In addition, we demonstrated that the intracellular processing after receptor binding plays an important role in determining the half-life of fusion proteins. While the binding of the GH domain to the GH receptor leads to endocytosis and lysosomal degradation in target cells, binding of the Tf domain to the Tf receptor may recycle the fusion protein and prolong its plasma half-life. To further confirm the effects of receptor binding on plasma half-life, G-CSF-Tf bifunctional fusion proteins with the 3 linkers were evaluated by administering the fusion proteins to the mice via intravenous administration. While the 3 fusion proteins showed a similar G-CSF receptor binding affinity, the G-CSF-Tf fusion protein with the higher Tf receptor binding affinity exhibited longer plasma half-life. This result further confirmed the involvement of Tf in recycling and prolonging plasma half-life. Based on our results, a mechanistic model was developed to summarize the crucial factors in determining the PK of the bifunctional fusion proteins. Our findings are useful for predicting the plasma half-lives, as well as for improving the pharmacokinetic profiles of therapeutic bifunctional fusion proteins by applying linker technology. ❧ Finally, to further extend our PK studies, the PK of Tf-fusion proteins via oral administration was investigated. A sandwich ELISA was developed to determine the serum level of G-CSF-Tf fusion proteins. A PK study of G-CSF-(H4)2-Tf fusion protein was conducted by orally dosing the mice. The results showed that intact G-CSF-(H4)₂-Tf fusion protein could be detected in the serum after oral dosage, and the serum level was physiologically significant. These results confirm our hypothesis that Tf-fusion proteins can be applied for oral delivery of protein drugs. By comparing the PK profiles of G-CSF-(H4)₂-Tf fusion protein after intravenous or oral administration, a shortened plasma half-life of the fusion protein was observed after oral administration. This result is consistent with our previously data for orally-absorbed Tf, which also exhibited shortened plasma half-life compared to intravenously administered Tf. This observation suggests that orally-absorbed Tf or Tf-fusion protein may have decreased stability, possibly due to the modifications on glycosylation or protein backbone during transcytosis. Another observation is that G-CSF-(H4)₂-Tf fusion protein exhibited faster absorption rate compared to Tf, indicating that the transcytosis process across intestinal epithelium cells might be different between Tf-fusion proteins and Tf. The establishment of the oral PK study can be applied for the investigation of other Tf-fusion proteins and for the improvement of the oral delivery system. ❧ In summary, the pharmacokinetics (PK) and pharmacodynamics (PD) of Tf-fusion proteins were investigated by using linkers as tools. An in vivo cleavable disulfide linker was designed for the release of free functional domains in vivo to achieved desired PK or PD properties. The PK of GH-Tf and G-CSF-Tf fusion proteins were investigated by linker insertion. As a result, the first mechanistic PK model was established for the bifunctional fusion proteins. The impact of linkers on PK of bifunctional fusion proteins was also demonstrated. Finally, the PK of G-CSF-(H4)₂-Tf fusion protein was studied via oral administration, and confirmed that Tf-fusion proteins can orally deliver protein drugs into the circulation.