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 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.

Keywords: G-CSF; PEGylated G-CSF; cancer chemotherapy; neutropenia; prolonged biological activity of G-CSF; site-specific PEGylation.

FIGURE 1

Structure assembly simulation of G-CSF using I-TASSER webserver. (A) The position of four lysine residues (at positions 16, 23, 34, and 40) in G-CSF structure by selection halos. (B) Cysteine position in the Cys2 variant and four indigenous cysteines involved in two disulfide bonds in the Cys2 G-CSF variant by selection halos. (C) The structure assembly simulation using the I-TASSER TM-align structural alignment program to compare the structures of the Cys2 variant (shown in multicolor ribbon cartoon) and human G-CSF (displayed in purple backbone trace). Helix A, B, C, and D are shown in blue, green, yellow, and red, respectively. Lysine and cysteine positions are depicted with yellow halos.

FIGURE 2

Purification and characterization of the G-CSF Cys2 variant. (A) Cation exchange chromatography profile of the Cys2 variant of G-CSF. Protein was eluted by 1 M Tris–HCl. Parameters such as absorbance at 280 nm and conductance have been represented with blue and brown lines, respectively. (B) The SDS-PAGE profile shows the general purity of the eluted Cys2 variant protein. (C) A comparison of far UV circular dichroic spectra of Cys2 cysteine variant and wild type G-CSF.

FIGURE 3

Purification and characterization of PEGylated Cys2 variant of G-CSF. (A) The first purification step (i.e., cation exchange chromatography, CEC) profile of the 20 kDa PEGylated Cys2 variant of G-CSF. (B) The second purification step (i.e., size exclusion chromatography, SEC) profile of CEC purified Cys2 variant protein in its PEGylated form. (C) Non-reducing SDS-PAGE profile stained with CBB showing the purity of purified PEGylated protein, wherein Lane 1 is the non-PEGylated cysteine variant, Lane 2 is the CEC-eluted peak fraction corresponding to PEG-conjugated Cys2 protein, Lane 3 is the SEC purified fraction, and Lane 4 is the Marker. (D) Same samples run on different gel and stained with barium iodide for staining specifically PEGylated protein. (E) The MALDI-TOF profile of purified Cys2 variant conjugated with 20 kDa PEG. (F) Comparison of far UV circular dichroic spectra of Cys2 variant conjugated with 20 kDa PEG and commercial available 20 kDa PEGylated G-CSF.

FIGURE 4

Biological activity of PEGylated variants in a neutropenia mice model. (A) Schematic depiction of the experimental procedure. CPA denotes cyclophosphamide. (B) Biological activity comparison of commercially available 20 kDa PEGylated G-CSF (standard) and Cys2 variant conjugated with 20 kDa, 30 kDa, and 40 kDa methoxy PEG maleimide in neutropenic mice. Total leucocytic counts (TLC) were determined following a single subcutaneous injection of 40 μg of G-CSF variants. The plots represent a scatter dot plot wherein data are means with SEM for three mice per group. Statistical significance was determined using two-way ANOVA multiple comparisons of the data. *indicates a P-value < 0.05, **indicates a P-value < 0.01 and ****indicates a P-value < 0.0001.

FIGURE 5

Characterization of PEGylated G-CSF Cys2 variant. (A) CBB-stained non-reducing SDS-PAGE analysis of purified higher-molecular-weight PEG-conjugated Cys2 variants. (B) Barium iodide-stained gel for purified protein samples to stain specifically PEGylated protein. (C) The MALDI-TOF profile of purified Cys2 variant conjugated to 30 kDa PEG and (D) 40 kDa PEG. (E) Comparison of far UV circular dichroic spectra of Cys2 variant conjugated with different sizes of PEG and commercially available 20 kDa PEGylated G-CSF.

FIGURE 6

Biological activity of half-dose and same-day administration of PEGylated variants. (A) Biological activity profile of half-dose of Cys2 variant conjugated with 40 kDa methoxy PEG maleimide in neutropenic mice. (B) Biological activity profile of Cys2 variant conjugated with 40 kDa methoxy PEG maleimide administered same-day (after 8 h of CPA treatment) and compared with the standard administered, i.e., after 24 h of induction of neutropenia in mice. The plots represent a scatter dot plot wherein data are means with SEM for three mice per group. Statistical significance was determined using two-way ANOVA multiple comparisons of the data. *indicates a P-value < 0.05, **indicates a P-value < 0.01, ***indicates a P-value < 0.001 and ****indicates a P-value < 0.0001.