Extension of human GCSF serum half-life by the fusion of albumin binding domain

Fatemeh Yadavar Nikravesh,Yeganeh Talebkhan,Samira Shirkhani,Hooman Aghamirza Moghim Aliabadi,Soroush Sardari,Elham Bayat,Yalda Hosseinzadeh Ardakani,Esmat Mirabzadeh,Masoumeh Sabzalinejad,Leila Nematollahi

doi:10.1038/s41598-021-04560-6

Fatemeh Yadavar Nikravesh, Yeganeh Talebkhan + Show 8 more

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https://doi.org/10.1038/s41598-021-04560-6

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Abstract

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Highlights

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts
In the present study, GCSF was genetically fused to the ABD094 domain to study the possible structural and functional changes of GCSF protein in comparison to the commercially available GCSF molecules
According to the endotoxin assessment by Pyrotell test, LPS content of the purified Albumin binding domain (ABD)-GCSF protein was lower than the detection limit of the kit (< 0.25EU/ml) when 2EU/mg LPS of the Filgrastim

Summary

Introduction

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties Inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1B) are being released early during the course of microbial infections. These factors stimulate NF-κB and C/EBPβ pathways which enhance granulocyte colony-stimulating factor (GCSF) expression[1] and higher production of granulocytes and stem cells from bone marrow. Further studies developed ABD molecules which were safer and had desirable affinity towards albumin (ABD088, ABD094 and ABD095)[29] Among these ABD derivatives, ABD094 showed the lowest sensitivity during T-cell proliferation a ssay[30]. In the present study, GCSF was genetically fused to the ABD094 domain to study the possible structural and functional changes of GCSF protein in comparison to the commercially available GCSF molecules

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