Protein engineering strategy to prolong the blood half-life of small-sized therapeutic proteins

Abstract

For the last decade, a number of therapeutic proteins have been developed and are now widely used in the clinic. They possess the broad spectrum starting from peptides, antibodies, cytokines, and hormones. A part from antibodies, most of these therapeutic proteins tend to have small molecular weight. Due to their small size, these therapeutic proteins result in fast clearance when injected into body and therefore show low therapeutic efficacy. This is because they undergo renal clearance by having molecular weight smaller than the renal filtration threshold, which is 60 kDa. As a result, their short half-life is considered as a critical drawback in the development of therapeutic agents.

Small-sized non-antibody scaffolds have also attracted considerable interest as alternatives to immunoglobulin antibodies. However, their short half-life is considered as a drawback in the development of therapeutic agents. We have demonstrated that a homo-dimeric form of a repebody enhances the anti-tumor activity than a monomeric form through prolonged blood circulation. Spytag and spycatcher were genetically fused to the C-terminus of a respective human IL-6-specific repebody, and the resulting two repebody constructs were mixed at an equimolar ratio to produce a homo-dimeric form through interaction between spytag and spycatcher. The homo-dimeric repebody was detected as a single band in the SDS-PAGE analysis with an expected molecular size (78 kDa), showing high stability and homogeneity. The dimeric repebody was shown to simultaneously accommodate two hIL-6 molecules, and its binding affinity for hIL-6 was estimated to be comparable to a monomeric repebody. The serum concentration of the dimeric repebody was observed to be about 5.5 times higher than a monomeric repebody, consequently leading to considerably higher tumor suppression effect in human tumor xenograft mice. The present approach can be effectively used for prolonging the blood half-life of small-sized protein binders, resulting in enhanced therapeutic efficacy.

Recombinant protein drugs derived from endogenous proteins show promising effects in variety of diseases. However, since most of these recombinant therapeutic proteins tend to have small molecular weight, they are likely to undergo fast clearance within several minutes when injected. We have demonstrated that a human serum albumin-specific repebody significantly prolong the blood circulation time of small-sized protein drugs and as a result enhance their therapeutic efficacy. Repebody that specifically binds with human serum albumin was selected through phage display. After one round of affinity maturation, the binding affinity was measured as 4.3 nM through surface plasmon resonance (SPR). The developed HSA-specific repebody showed high binding stability and enhanced pharmacokinetics profile. The terminal half-life was 6.7 h and area under curve value was 12.3-fold higher compared to off-target repebody. Glucagon-like Peptide-1 (GLP-1) with DPP-VI resistance was genetically fused to the C-terminus of an HSA-specific repebody as a proof of our platform. The repebody-fused GLP-1 was expressed in E. coli with high purity and yield, confirmed by size exclusion chromatography and SDS-PAGE analysis. The GLP-1 fused with HSA-specific repebody showed prolonged blood circulation profile compared fusion with off-target repebody resulting in a terminal half-life of 10.2 h and 4.2-fold increase of area under curve value. As a result, the HSA-specific repebody-fused GLP-1 significantly enhanced control over blood glucose level in both normal and type 2 diabetes model mice compared to both wild type and off-target repebody-fused GLP-1. This HSA-specific repebody based therapeutic protein delivery platform can be effectively and generally used for prolonging the blood half-life of various small-sized protein therapeutics, and hence result in enhanced therapeutic efficacy.

In this thesis, we will demonstrate various engineering strategies to elongate the blood circulation time of these small-sized therapeutic proteins, especially by utilizing protein dimerization and human serum albumin. The proposed strategies can be generally used for the elongation of blood circulation time of small-sized therapeutic proteins, and hence result in enhanced therapeutic efficacy.