Developing Long-acting Biotherapeutics:Site-selective PEGylation And Fusion To Albumin-binding Domain

Various half-life extension strategies have been developed and applied on biotherapeutic proteins to improve their pharmacokinetics.PEGylation of drug protein is able to extend the protein's half-life,to reduce their innate immunogenicity and improve the physiochemical stability and water-solubility.Coupling polyethylene glycol with biotherapeutics increases proteins' hydraulic radius and thus reduce glomerular filtration to improve their pharmacokinetics.Another widely-used approach is based on neonatal Fc receptor(FcRn)mediated cell recycling.Here in this dissertation,we developed a new thiol-selective PEGylation reagent and,extended the application of albumin-binding domain fusion strategy on CNTF,as well as compared the two strategies.Thiol-specific PEGylation of clinically used protein via free cysteine residue has been widely applied for a homogeneous product.However,limitations have been reported to market-available reagents,such as the succinimide ring is susceptible to ring-opening hydrolysis,lower reactivity or de-conjugation of the protein from the PEG.Herein,we developed a new thiol-specific PEGylation reagent based on catechol-derived reactive quinone species.Catechol-derived polyethylene glycol(PEG)was synthesized by coupling linear PEG N-hydroxysuccinimide to dopamine and then oxidized to quinone.First,PEG-dopaquinone(PEG-DAQ)mostly modifying the free cysteines of three model proteins of truncated flagellin(CBLB502)and recombined human ciliary neurotrophic factor(CNTF),BSA evidenced its thiol-reactivity and thiol-specificity.Furthermore,PEG-DAQ coupling efficiency were compared with another two available reagents PEG-MAL or PEG-VS.The yield of PEG-DAQ-CNTF under neutral condition was 87.5%,compared with PEG-maleimide of 92.3%and PEG-vinylsulfone of 17.6%,respectively.Moreover,the effect of pH on PEGylation efficiency was investigated to show that the bioconjugation occurred at various pH(pH 5.5-8.5),and the PEGylation efficiency increased with higher pH.About 95%reactive capacity for PEG-DAQ was maintained after incubation in aqueous solvent for 96 hours,indicating that the reagent was superiorly hydro-stable;whereas,approximately 70%of the initial reactivity of PEG-MAL was remained.Furthermore,the application of PEG-DAQ reagents was investigated using CNTF as model protein.Mono-PEGylated CNTF has been prepared through bioconjugation and further purification.Circular dichroism analysis and fluorescence spectra were measured to analyze the secondary and tertiary structure of CNTF and PEGylated CNTF.The data indicated that PEGylated CNTF showed similar secondary and tertiary structure as the native protein.Moreover,the stability of the PEGylated species via PEG-MAL and PEG-DAQ were compared.It was found that PEGylation reaction with excess cysteine quenching was necessary to produce more stable conjugates.The resulted PEG-DAQ-CNTF remained about 93.5%of the initial products after storage for 14 days.As a comparison,PEG-MAL-CNTF was remained only 71.2%.Finally,the CCK-8 analysis of cell viability demonstrated its low cell toxicity and the pharmacokinetic test suggested PEG-DAQ could extend CNTF's half-life by three times.Our results revealed that PEG-dopaquinone had properties of high selectivity and reactivity with cysteine thiol,and superior stability for both the reagent itself and its conjugate,showing great promise for developing as an alternative reagent for thiol-selective modification.Human serum albumin(HSA)is one of most abundant protein in the serum.HAS generally exhibit a circulatory half-life of 19 days due to its large molecular size and neonatal Fc receptor(FcRn)mediated cell recycling.Fusion with endogenous serum albumin as a drug carrier is another increasingly attractive strategy to extend the circulatory time for small protein and peptide.This fusion strategy avoiding chemical modification and further purification.However,production of these fused large proteins generally requires mammalian cells,a relatively complicated and expensive host system,always leading to lower expression and higher cost.Albumin-binding domain(ABD)is a small peptide,which can bind with endogenous HSA and further utilize the naturally long-half-life property of HSA.Herein we constructed a new long-acting CNTF by genetic fusion with an albumin-binding domain(ABD)through a flexible peptide linker,and compared the ABD fusion strategy with the established PEGylation approach.This fused protein ABD-CNTF was expressed in E.coli mainly in the soluble form and purified through a two-step chromatography,with purity of 95%and a high yield of 90-100 mg/L culture.The in-vitro binding ability of ABD-CNTF with HSA was verified by incubation of the two components together followed by HP-SEC analysis.ABD-fused CNTF showed similar secondary and tertiary structure as the native protein.It retained approximately 94.1%of the native bioactivity,whereas PEGylated CNTF suffered severe activity loss,as demonstrated via CCK-8 cell viability assay analysis.In-vivo studies in SD rats were performed and the terminal half-life of ABD-CNTF was determined to be 483.89 min,which is about 14 folds longer than that of native CNTF(34.28min)and is comparable with 20k-40 kDa PEGylated CNTFs.The new constructed ABD-CNTF represents a potential therapeutic modality.The results suggested the ABD-fusion is a cost effective and efficient strategy for long-lasting biopharmaceutics' design.