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

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 sitespecific 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.

Optimizing Granulocyte Colony-Stimulating Factor Transcript for Enhanced Expression in Escherichia coli

“…The details of all chemicals, molecular biology reagents, G-CSF standard, protein purification resins, columns, etc., were reported in Kumari et al (2020). Protein concentrators were procured from Merck Millipore.…”

“…The human G-CSF cDNA, codon-optimized gene sequence and cloning details are as provided earlier ( Kumari et al, 2020 ). Briefly, the native human G-CSF cDNA sequence was PCR amplified using forward primer SD13 and reverse primer SD14 and cloned in pET 23a.…”

“…After centrifugation, the supernatant (soluble fraction) and the pellet (insoluble fraction) were analyzed using 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue. G-CSF was purified using the method reported earlier in Kumari et al (2020) . Briefly, the G-CSF protein was expressed as inclusion bodies, which were solubilized using 2 M urea.…”

“…The intact molecular mass of G-CSF and the control was determined using liquid chromatography-mass spectrometry (LC-MS) Agilent 6550 system. Circular dichroism (CD) spectrometry was performed, as mentioned earlier on the Jasco J-815 spectropolarimeter ( Kumari et al, 2020 ).…”

“…The human G-CSF cDNA, codon-optimized gene sequence and cloning details are as provided earlier (Kumari et al, 2020).…”