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Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats

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Abstract

Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

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Abbreviations

CBC:

Complete blood cell count

CPA:

Cyclophosphamide

EC50 :

Concentration causing half maximal response

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

G-CSF:

Granulocyte colony-stimulating factor

GM-CSF:

Granulocyte-macrophage colony-stimulating factor

Hu:

Human

IV:

Intravenous

Mu:

Murine

PEG:

Polyethylene glycol

RT-PCR:

Reverse transcriptase-polymerase chain reaction

SC:

Subcutaneous

SD:

Standard deviation

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM:

Standard error of the mean

TCEP:

Tris[2-carboxyethylphosphine]hydrochloride

WBC:

White blood cell (total of lymphocytes, neutrophils, monocytes, eosinophils, and basophils)

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Acknowledgements

We thank Jennifer Hughes and Sharon Carlson for performing the ELISA and WinNonlin pharmacokinetic analyses.

Funding

This research was supported by Grant R44CA084850 from the National Cancer Institute to DHD. The publication’s contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute or The National Institutes of Health.

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Authors and Affiliations

  1. Bolder BioTechnology, Inc., 2425 55th Street, Suite 210, Boulder, CO, 80301, USA

    George N. Cox, Ji I. Lee, Mary S. Rosendahl & Daniel H. Doherty

  2. Premier Laboratory, LLC, 1567 Skyway Drive, Unit E, Longmont, CO, 80504, USA

    Elizabeth A. Chlipala

Authors
  1. George N. Cox
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  2. Ji I. Lee
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  3. Mary S. Rosendahl
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  4. Elizabeth A. Chlipala
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  5. Daniel H. Doherty
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Corresponding author

Correspondence to George N. Cox.

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Conflict of interest

GNC, DHD, MSR, and JIL are employees or former employees of Bolder BioTechnology. GNC and DHD have a financial interest in Bolder BioTechnology. GNC, DHD, and MSR are inventors on patents and patent applications owned by Bolder BioTechnology related to GM-CSF and other proteins and methods. EAC has a financial interest in Premier Laboratory, which was paid by Bolder BioTechnology to perform the animal studies described in the manuscript, as well as other animal studies.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of Premier Laboratory and the University of Colorado, Boulder, where the studies were conducted.

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Cox, G.N., Lee, J.I., Rosendahl, M.S. et al. Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats. Protein J 39, 160–173 (2020). https://doi.org/10.1007/s10930-020-09894-0

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