Development of Site-Specific PEGylated Granulocyte Colony Stimulating Factor With Prolonged Biological Activity.

Monika Kumari,Sonal Datta,Girish Sahni

doi:10.3389/fbioe.2020.572077

Abstract

Currently, amino-terminal PEGylated human granulocyte colony stimulating factor (huG-CSF) is used to prevent and treat neutropenia. Although huG-CSF has been used as a drug for more than 20 years, it has three significant drawbacks: (i) it relies on PEG aldehyde for PEGylation of the alpha-amino group of the first amino acid, and this leads to non-specific PEGylation of the epsilon amino group of lysine residues within the G-CSF; (ii) longer-acting G-CSF variants are desirable to reduce the risk of chemotherapy-associated neutropenia; and (iii) G-CSF cannot be administered on the day of chemotherapy. In an attempt to overcome the above drawbacks, we engineered cysteine variants of G-CSF to facilitate the maleimide PEG-based site-specific PEGylation that leads to a highly homogenous PEGylated product. Importantly, we have demonstrated that 20 kDa thiol-reactive PEG conjugated by maleimide chemistry to the Cys2 G-CSF variant exhibits leukocyte proliferative activity similar to that of the commercially available G-CSF conjugated with aldehyde PEG in a neutropenia mice model. Moreover, we have demonstrated that PEGylation of the cysteine variant of huG-CSF with higher molecular weight PEGs, such as 30 kDa PEG and 40 kDa PEG, leads to significantly prolonged leukocyte proliferation activity compared to the variant conjugated with 20 kDa PEG. Importantly, even a half-dose of the engineered variant conjugated with 40 kDa PEG exhibited significantly longer biological activity than the commercially available 20 kDa PEGylated huG-CSF. Finally, we have demonstrated that administration of the engineered variant conjugated with 40 kDa PEG on the day of administration of cyclophosphamide for inducing neutropenia in mice can alleviate neutropenia through leukocyte proliferation. In summary, this study provides the design of site-specific PEGylated huG-CSF variants with improved therapeutic potential. It opens the possibility of long-acting and same-day prophylactic administration of G-CSF after chemotherapy drug regimens. These results may pave the way for the development of potential G-CSF derivatives possessing longer half-lives and favorable clinical attributes.

Highlights

Human granulocyte colony stimulating factor is a 19 kDa cytokine that stimulates the proliferation, maturation, and functional activation of the cells in the granulocyte lineage (Anderlini et al, 1996; Welte et al, 1996)
We utilized the TM-align structural alignment program to compare G-CSF Cys2 I-TASSER simulation to all the structures in the PDB library, which confirmed the substitution of cysteine at position 2 does not affect the salient structural features of the G-CSF (Figure 1C)
We have successfully designed G-CSF variants for site-specific PEGylation, which may provide a solution toward prolonged half-life and biological activity

Summary

Introduction

Human granulocyte colony stimulating factor (huG-CSF) is a 19 kDa cytokine that stimulates the proliferation, maturation, and functional activation of the cells in the granulocyte lineage (Anderlini et al, 1996; Welte et al, 1996). G-CSF is cleared from the human body through various means, including receptormediated endocytosis followed by its degradation (Kuwabara et al, 1996), renal clearance (Kuwabara et al, 1995), and enzymatic degradation mechanism (El Ouriaghli et al, 2003). G-CSF has a short circulation half-life of about 3.5 h (Ng and Tang, 2013). This necessitates the administration of daily injections to maintain the effective concentration in the body to combat neutropenia

Results

Discussion

Conclusion